Post-incision events induced by UV : regulation of incision and the role of post-incision factors in mammalian NER
Overmeer, R.M.
Citation
Overmeer, R. M. (2010, September 29). Post-incision events induced by UV : regulation of incision and the role of post-incision factors in mammalian NER.
Retrieved from https://hdl.handle.net/1887/15997
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Summary for the layman
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Summary for the layman
DNA has become a popular subject for advertising, in part due to the mysterious promise of miracle cures held within its double helix. It is this double helix which gives DNA its iconic shape and enables it to perform its main function; ensuring accurate duplication and transmission of information to daughter cells. The helical structures usually depicted actu- ally represent the backbone of DNA, with the basepairs being the ladder connecting the two strands. There are four different bases which each have a specifi c complementary partner, as such each adenine (A) is opposite to a thymine (T) and each cytosine (C) being opposite to a guanine (G) and vice versa (i.e. T-A and G-C). The order of the bases encodes the information required for a cell to function. A single human cell contains 46 chromosomes, each being between 48 and 250 million basepairs in length and as such the amount of information stored in our DNA. This complete complement of genetic information contained within each cell is called the genome.
To duplicate the DNA (i.e. DNA replication) the strands are fi rst pulled apart and new bases are then placed complementary to the original base, forming new basepairs (with A op- posite a T and C opposite to a G). Each base is connected to the previous one prior to the next base aligning with the original strand and two new double strands are formed, one for each daughter cell. One can imagine that the integrity of the backbone is essential for this process, for if the backbone is broken DNA replication would stall and the base sequence would be compromised and the information lost. Similarly the integrity of the bases is equally essential as when the base is unable to for a pair with its opposite base replication would similarly stall and/or wrong bases could be inserted, which would also lead to a loss or corruption of information.
Our DNA is constantly subject to damage by both environmental factors such as tobacco smoke, radiation and sunlight (in the form of UV irradiation) but also our own metabolism (i.e. the conversion of food into energy) creates radical oxygen species which damage our DNA. To counteract this continuous assault and ensure integrity of our genome, cells pos- ses a number repair pathways on one hand and can prevent the progression of cellcycle (the process of cell division) on the other. Should a cell progress with the cellcycle with damage in the genome information can be lost or changed. Such changes in the genome can lead to inherited diseases and cancer.
The focus of this research is on the major repair pathway after UV irradiation and the signalling pathway induced after UV irradiation. More specifi cally this thesis focuses on these events in non-dividing cells. This distinction is important as many of the factors in- volved in repair are also involved in DNA replication which would therefore interfere with our measurements.
The fi rst chapter describes the aim of this thesis and briefl y describes the various types of damage and associated repair processes. Furthermore the inherited diseases associated with defi cient nucleotide excision repair (NER) are described. Subsequently NER is described in
more detail as this is the main pathway involved in repair of UV induced DNA damage. In short NER repairs UV induced damage by removing a single strand of DNA of approxima- tely 30 basepair in length. The resulting single stranded gap is then fi lled in a similar fashion as normal DNA replication. The recognition of DNA damage by NER can be divided into two subpathways; transcription coupled NER (TC-NER) and global genome NER (GG-NER). As the main topic of this thesis is GG-NER, chapter 2 describes the recognition of DNA damage and assembly of GG-NER in depth. In addition the transition from the incision complex to the gapfi lling complex, which are two distinct complexes, is discussed. Moreover the regu- lation of NER is also discussed. Chapter 2 also discusses the dynamic nature of the proteins involved in NER and the signalling cascade induced by UV irradiation in non-cycling cells.
The perspectives granted by recent advancements, in respect to this thesis, are discussed in chapter 3.
The fourth chapter describes the regulation of NER, a process which was observed fi rst more that 2 decades ago, yet remained elusive until recently. To study this regulation a novel method, based on a series of (local) UV irradiations, was developed. By allowing complexes to accumulate at initial damages prior to inducing a second set of damages, the dissociation of proteins from the initial damages could be observed. Furthermore by doing these experi- ments under conditions where repair was unable to be completed, the dissociation of proteins from sites of incomplete repair could be described. These observations led to the observation that RPA, which is required for incision and is also involved in gapfi lling, is the central player in regulating incision as its accumulation in post-incision (gapfi lling) complexes prevents its association with pre-incision complexes, which are subsequently unable to incise and remove the damaged DNA.
As mentioned, after incision the resulting gap of single stranded DNA must be fi lled in a manner similar to normal replication. An essential part of this repair replication is the recruitment and loading of a ring-shaped platform called a sliding clamp (PCNA) by a clamp loader. This sliding clamp then acts as a mobile platform binding the DNA polymerase(s), enabling DNA replication by correctly positioning the polymerase(s). Chapter 5 describes the role of the clamploader, required for normal DNA replication (i.e. RFC), in NER. The results show that the clamploader (RFC) is not required for the recruitment of the clamp (PCNA) but is required for the recruitment of the DNA polymerase δ. These results imply that PCNA recruitment is independent of RFC and that either the loading of PCNA by RFC is required for recruitment of DNA polymerase δ or that RFC directly recruits DNA polymerase δ.
DNA polymerase δ is not the only polymerase available, there are numerous DNA po- lymerases in the cell, each with their own activity and fi delity. In addition to the high fi delity polymerases δ and ε there are more promiscuous polymerases such as κ, η and ι which are involved in translesion synthesis, an error prone process or replicating past base damage to ensure that DNA replication is completed (incomplete replication generally carries more severe consequences than single base changes). The involvement of these promiscuous poly- meares in NER is described in chapter 6. Surprisingly the gapfi lling seems to be performed
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by two different pathways, each with their own distinct composition of polymerases and clamploaders.
As mentioned, it is important for cells to control cellcycle progression in the presence of damage to ensure that the damage is repaired before continuing to the next phase and ultimately division. As discussed in chapter 2 DNA damage signalling is usually induced by recognition of repair intermediates as opposed to the damage itself. Chapter 7 describes the activation of signalling in NER defi cient cells. Interestingly, Ape1, a protein usually involved in a separate repair process (base excision repair or BER), incises at UV induced damages.
However this incision leads to a structure that cannot be repaired by BER as this process removes single bases and UV damage induces dimerisation of adjacent bases (any combi- nation of Cs and/or Ts). Although this intermediate cannot be repaired, it is recognized by the damage signalling pathway and, as such, prevents cells from progressing to the S-phase (where DNA replication takes place).
In conclusion little was known about the regulation of incision, the handover from pre- to post-incision and the exact requirement for gapfi lling. In addition to (partially) answering these questions this thesis also describes a novel method of activating damage signalling in NER defi cient cells.
